Chalk, A. M. | Search bioRxiv

bioRxiv2025

Minute amounts of helicase-deficient truncated RECQL4 are sufficient for DNA replication.

RECQL4 is a member of the RecQ family of helicases, playing essential roles in DNA replication and maintaining genome integrity. Mutations in RECQL4 are linked to severe human diseases, including Rothmund-Thomson Syndrome, RAPIDALINO Syndrome, and Baller-Gerold Syndrome. However, we still do not fully understand its functions and genetic interactions. The role of the ATP-dependent helicase activity in RECQL4 remains controversial. To understand RECQL4s functions further, we conducted a genome-wide forward genetic screen using murine models that closely mimic the RECQL4 mutations found in patients with Rothmund-Thomson syndrome. Our goal was to identify loss-of-function alleles that could rescue the proliferation and viability defects associated with RECQL4 mutation. From our screening we identified the loss of KLHDC3, a substrate-binding subunit of the Cullin-RING ligase (CRL) E3, as the most significant rescue allele. KLHDC3 facilitates the ubiquitin-mediated destruction of proteins with specific C-terminal degron motifs. Its loss normalized cell proliferation and DNA replication rates in cells with mutated RECQL4. Further analysis revealed that the loss of KLHDC3 led to the stabilization of minute levels of a truncated RECQL4 protein. This RECQL4 fragment contained a neo-degron sequence specific for KLHDC3, formed after Cre-mediated recombination of the Recql4fl allele. Although this rescue mechanism does not apply to human RECQL4 mutations, it shows that very low chromatin-bound levels of a truncated RECQL4 protein--comprising only the N-terminal 480 amino acids, including its Sld2-like domain but lacking the ATP-dependent helicase domain and the entire C-terminal portion--are sufficient to support DNA replication in mammalian cells. These results demonstrate that the ATPase activity and helicase domain of RECQL4 are not essential for DNA replication in mammals. Furthermore, our findings suggest that there are unlikely to be monogenic loss-of-function alleles that can rescue RECQL4 mutations. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require the ATP dependent helicase activity.

cell biology↗

bioRxiv2022

Srsf2P95H/+ co-operates with loss of TET2 to promote myeloid bias and initiate a chronic myelomonocytic leukemia like disease in mice.

Recurrent mutations in two pathways - the RNA spliceosome (eg. SRSF2, SF3B1, U2AF1) and epigenetic regulators (eg. DNMT3, TET2) - contribute to the development of myelodysplastic syndrome (MDS) and related myeloid neoplasms. In chronic myelomonocytic leukemia (CMML), SRSF2 mutations occur in [~]50% of patients and TET2 mutations in [~]60%, representing two of the most frequent mutations in these cancers. Clonal analysis has indicated that either mutation can arise as the founder lesion, however, our understanding of the basis for the co-operativity of these mutations in the evolution of CMML is limited. Based on human cancer genetics we crossed an inducible Srsf2P95H/+ mutant model with Tet2fl/fl mice to mutate both concomitantly (or individually) in hematopoietic stem cells. At 20-24 weeks post gene mutation, we observed subtle differences in the Srsf2/Tet2 mutants compared to either single mutant. Under conditions of native hematopoiesis with aging, we see a distinct myeloid bias and monocytosis in the Srsf2/Tet2 mutants. A subset of the compound Srsf2/Tet2 mutants display an increased granulocytic and distinctive monocytic proliferation (myelo-monocytic hyperplasia), with increased immature promonocytes and monoblasts ([~]10-15% total nucleated cells), and evidence of binucleate promonocytes. Exome analysis of progressed disease demonstrates mutations in genes and pathways similar to those reported in human CMML. Upon transplantation, recipients developed leukocytosis, monocytosis and splenomegaly. This demonstrates we can reproduce Srsf2/Tet2 co-operativity in vivo, yielding a disease with core characteristics of CMML, unlike single Srsf2 or Tet2 mutation. This model represents a significant step toward building high fidelity and genetically tractable models of CMML. Key pointsO_LISrsf2P95H/+ co-operates with Tet2-/- to initiate CMML in a murine model C_LIO_LISrsf2P95H and Tet2 null mutations synergize in the development of monocytosis C_LI

cancer biology↗